Renal regulatory elements and methods of use thereof

ABSTRACT

Disclosed are cis-acting regulatory elements from a KIM-1 gene. The elements can be used to direct the expression of operably linked sequences in renal tissue.

STATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with federal government support undergrant #DK 39773. The United States government has certain rights in theinvention.

FIELD OF THE INVENTION

[0002] The invention relates generally to nucleic acids and moreparticularly to nucleic acids which can be used to direct expression inrenal tissue of operably linked sequences.

BACKGROUND OF THE INVENTION

[0003] Significant interruption in kidney function in an individual canlead to incapacitation or even death. Disease or injury can impairkidney function. An example of an injury that can damage kidneys isischemic injury. In this type of injury, kidney tissue is damagedbecause of oxygen deprivation occurring as a result of interruption ofblood flow to the kidneys.

[0004] Certain agents involved in repair of diseased or damaged kidneytissue, and mechanisms in which the repair takes place, have beendescribed. Mechanisms include induction of gene expression andrecruitment of growth factors to the affected kidney tissue. Cell deathand cellular proliferation are also associated with repair or kidneytissue.

[0005] Agents implicated in kidney tissue repair include polypeptides,e.g., growth factors such as insulin growth factor (IGF), epidermalgrowth factor (EGF), hepatocyte growth factor (HGF), and the endothelialcell adhesion molecule ICAM-1.

[0006] Recently, the polypeptide kidney-injury molecule (KIM-1) has beendescribed. The expression of KIM-1 is increased in injured kidneytissue. The rat and human forms of this protein have been characterized.The KIM-1 cDNA sequence reveals that the KIM-1 protein is a type 1membrane protein that contains a novel six-cysteine immunoglobulin-likedomain and a mucin domain. The KIM-1 protein is a member of theimmunoglobulin gene superfamily and most closely resembles mucosaladdressin cell adhesion molecule 1 (MAdCAM-1).

SUMMARY OF THE INVENTION

[0007] It has been discovered that nucleic acid sequences in thevicinity of the human KIM-1 gene can be used to express linked sequencesin renal tissue. Accordingly, the invention provides a cis-actingregulatory element useful for, inter alia, directing expression of anoperatively linked sequence, e.g., a gene, in a mammal. The cis-actingKIM-1 regulatory sequence can also be used to identify trans-actingfactors that mediate the response of the kidney to damaged or diseasedtissue.

[0008] The invention provides an isolated nucleic acid that includes acis-acting KIM-1 derived regulatory sequence. The nucleic acid can be,e.g., a nucleic acid sequence that includes the nucleic acid sequence ofSEQ ID NOs:1, 2 or 3. The nucleic acid includes at least 5 contiguousnucleotides from a sequence that hybridizes to SEQ ID NOs: 1, 2, or 3,or sequences complementary to SEQ ID NOs: 1, 2, or 3. For example theregulatory sequence can include between 5 and 35 contiguous nucleotidesfrom SEQ ID NO:3, or sequences complementary to such portions of SEQ IDNO:3.

[0009] In some embodiments, a cis-acting KIM-1 regulatory sequenceaccording to the invention includes a portion of SEQ ID NOs:1, 2 or 3sufficient to regulate kidney tissue-specific transcription of anoperably linked sequence, e.g., an operably linked polypeptide-encodingsequence. A cis-acting KIM-1 regulatory sequence according to theinvention can include a portion of SEQ ID NOs:1, 2 or 3 is sufficient toregulate kidney tissue-specific transcription following cellular injurye.g., anoxia or exposure to reactive oxygen species (“ROS”), or in acell present in a confluent population of cells.

[0010] The invention also provides a cis-acting KIM-1 regulatorysequence operably linked to a sequence encoding a KIM-1 antisensenucleic acid. The cis-acting KIM-1 regulatory sequence can be operablylinked to at least one polypeptide-encoding sequence and regulates renaltissue-specific transcription of the polypeptide-encoding sequence. Forexample, the polypeptide-encoding sequence may encode a KIM-1polypeptide (e.g., a human KIM-1 polypeptide), or a non-KIM-1polypeptide. This polypeptide can be, e.g., a cell survival-promotingfactor, a cell growth-promoting factor, a wound-healing factor, ananti-fibrotic factor, an apoptosis-inhibiting factor, ananti-inflammatory factor, a terminal differentiation-promoting factor, acell growth-inhibiting factor, an intravascular-volume restorationfactor, a chelating agent, an alkylating agent, anangiotensin-converting enzyme-inhibiting factor, erythropoietin, acytokine, a receptor, an anticoagulant, an enzyme, a hormone, anantibody, or a renal structural protein.

[0011] A cis-acting KIM-1 regulatory sequence according to the inventionmay be linked to, e.g., nucleic acid sequences encoding insulin growthfactor (IGF), an epidermal growth factor (EGF), a fibroblast growthfactor (FGF), a transforming growth factor beta (TGF β) Type IIreceptor, a hepatocyte growth factor (HGF), or an endothelial celladhesion molecule ICAM-1.

[0012] The invention also provides a vector that includes a nucleic acidcomprising a cis-acting KIM-1 regulatory sequence and cells containingthese nucleic acids and vectors. The cell can be prokaryotic oreukaryotic. The cell can be, e.g., a metazoan organism or a unicellularorganism, and can include, e.g., a fungal cell, yeast cell (such asSaccharomyces, Schizosaccharomyces, or Candida spp.), or a mammaliancell, e.g., a human, canine, bovine, porcine, feline, or rodent cell, ora non-human mammalian embryonic blastocyst cell.

[0013] The invention also provides a transgenic non-human mammal, e.g.,a mouse, rat, goat, pig, cow, or sheep, containing an isolatedcis-acting KIM1 regulatory sequence. The transgenic animal can beproduced, e.g., by intrauterine implantation of a blastocyte cellcontaining a cis-acting KIM-1 regulatory sequence. The invention alsoincludes one or more progeny of the transgenic non-human mammal DNA,wherein the progeny comprises the cis-acting DNA, or a fragment thereof.

[0014] The invention also provides a method of directing expression of apolypeptide. The method includes providing a cell, e.g., a renal cell,that includes an isolated cis-acting KIM-1 regulatory sequence operablylinked to sequence encoding a polypeptide of interest, culturing thecell under conditions that allow for the expression of the polypeptideand expressing the polypeptide-encoding sequence.

[0015] The invention also includes a method of increasing transcriptionof a polypeptide-encoding sequence in tissue. The method includesproviding a cell in the tissue that includes a cis-acting KIM-1regulatory sequence linked to the polypeptide-encoding sequence andculturing the cell under conditions that allow for the transcription ofthe polypeptide-encoding sequence. The polypeptide-encoding sequence isthen expressed, resulting in transcription of the polypeptide-encodingsequence in the tissue.

[0016] The invention also includes a method for identifying a testcompound that modulates expression from a cis-acting KIM-1 derivedregulatory sequence. The test compound can be contacted with a reporterconstruct that includes a reporter gene operably linked to an isolatedcis-acting KIM-1 regulatory sequence. The level of expression of thereporter gene in the tissue is detected, e.g., measured. A change in thelevel of expression in the presence of the test compound relative to thelevel of expression in the absence of the test compound indicates thatthe test compound modulates the activity of the KIM promoter.

[0017] The invention also provides a method for delivering a therapeuticpolypeptide to renal tissue of a subject. The method includes providingin the renal tissue a cell that includes a cis-acting KIM-1 regulatorysequence operably linked to a therapeutic polypeptide, and culturing thecell under conditions that allow for the expression of the polypeptide.The polypeptide-encoding sequence is expressed, thereby delivering thetherapeutic polypeptide to the renal tissue of the subject.

[0018] The invention also includes a method for treating or preventingrenal tissue injury. The method includes providing a cell that includescis-acting KIM-1 regulatory sequence operably linked to a polypeptidecoding sequence, and culturing the cell under conditions that allow forthe expression of a therapeutic polypeptide-encoding sequence. Thetherapeutic polypeptide-encoding sequence is expressed, and theexpressed polypeptide contacts the renal tissue, thereby treating orpreventing renal tissue injury.

[0019] The invention also includes a method for increasing transcriptionof a nucleic acid in a subject by administering to the subject acis-acting KIM-1 regulatory sequence operably linked to the nucleic acidand allowing for expression of the operably linked nucleic acid.

[0020] The invention also provides a method for treating or preventingrenal tissue injury in a subject by administering to a subject in needthereof a cis-acting KIM-1 regulatory sequence operably linked to asequence encoding a therapeutic polypeptide, in an amount sufficient totreat or prevent renal tissue injury in the subject.

[0021] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0022] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGS. 1A-H are schematic representations of a nucleic acidsequence that includes a cis-acting KIM-1 regulatory sequence (SEQ IDNO:1).

[0024] FIGS. 2A-D are schematic representations of sequences from the 5′region of the human KIM-1 gene.

[0025]FIG. 3 is a bar graph showing relative expression of luciferase inCOS monkey kidney cells of various reporter constructs containingsequences from the 5′ region of the human KIM-1 gene

[0026]FIG. 4 is a bar graph showing relative expression of luciferase inLLCPK cells of various reporter constructs containing sequences from the5′ region of the human KIM-1 gene.

[0027]FIG. 5 is a bar graph showing relative expression of luciferase inMDCK cells of various reporter constructs containing sequences from the5′ region of the human KIM-1 gene.

[0028]FIG. 6 is a bar graph showing inducibility of sequences linked toa KIM-1 regulatory sequence in HK2 cells after exposure to reactiveoxygen species (ROS) or anoxia.

[0029]FIG. 7 is a bar graph showing relative expression of luciferase inMDCK cells at various timepoints after transfection with reporterconstructs containing sequences from the 5′ region of the human KIM-1gene.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The invention provides a cis-acting regulatory element usefulfor, inter alia, directing expression of an operatively linked sequence,e.g., a gene, in a mammal. This cis-acting regulatory sequence fromKIM-1 can also be used to identify trans-acting factors that mediate theresponse of the kidney to damaged or diseased tissue.

[0031] Sequence Identifier Numbers (SEQ ID NOs)

[0032] Sequence identifier numbers used herein include the following:

[0033] SEQ ID NO:1 corresponds to the nucleotide sequence of an 8933 bphuman genomic DNA from the 5′ region of KIM-1 gene and is disclosed inFIGS. 1A-H. This fragment is present as a BamH1-BamH1 insert in theBamHI site of the EMBL3 phage vector. The construct is named MZ007.

[0034] SEQ ID NO:2 corresponds to the nucleotide sequence of a 4.8 kbKpnI-KpnI fragment encompassing nucleotides 3796 to 8612 of the humanKIM-1 insert in MZ007.

[0035] SEQ ID NO:3 corresponds to the nucleotide sequence of a 1.3 kbEcoR1-KpnI fragment encompassing nucleotides 7322-8612 of human KIM-1insert in MZ007.

[0036] SEQ ID NO:4 corresponds to the nucleotide sequence of a 0.5 kbSacII-KpnI fragment encompassing nucleotides 8110-8612 of human KIM-1insert in MZ007.

[0037] Cis-Acting KIM-1 Derived Regulatory Sequences

[0038] Included in the invention is an isolated DNA that includes acis-acting KIM-1 derived regulatory sequence. The term “isolated” refersto molecules separated from other DNA or RNA molecules, present in thenatural source of the regulatory sequence. The term also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. An isolated nucleic acid includes nucleic acid fragmentsthat are not naturally occurring as fragments and would not be found inthe natural state. The term “isolated” is also used herein to refer topolypeptides that are isolated from other cellular proteins, and theterm is meant to encompass both purified and recombinant polypeptides.

[0039] A cis-acting KIM-1 derived regulatory sequence, also termedherein “cis-acting regulatory element”, “regulatory element”, or“regulatory sequence”, includes nucleic acid sequence elements derivedfrom sequences in the vicinity of a mammalian KIM-1 gene that arecapable of modulating transcription from a basic promoter, as well asenhancers or silencers. The terms “promoter” and “regulatory element”further encompass “tissue specific” promoters and regulatory elements,i.e., promoters and regulatory elements which bring about expression ofan operably linked DNA sequence preferentially in specific cells (e.g.,cells of a renal tissue). Gene expression occurs preferentially in aspecific cell if expression in this cell type is significantly higherthan expression in other cell types. The terms “promoter” and“regulatory element” also encompass so-called “leaky” promoters and“regulatory elements”, which regulate expression of a selected DNAprimarily in one tissue, but cause expression in other tissues as well.The terms “promoter” and “regulatory element” also encompass non-tissuespecific promoters and regulatory elements, i.e., promoters andregulatory elements which are active in most cell types.

[0040] A promoter or regulatory element can be a constitutive promoteror regulatory element, i.e., a promoter or regulatory element whichconstitutively regulates transcription, or it can be a promoter orregulatory element which is inducible, i.e., a promoter or regulatoryelement which is active primarily in response to a stimulus. A stimuluscan be, e.g., a physical stimulus, such as injury (e.g., ischemia),and/or a molecule, such as a hormone, a cytokine, a heavy metal, phorbolesters, cyclic AMP (cAMP), or retinoic acid.

[0041] The term “enhancer”, also referred to herein as “enhancerelement”, includes regulatory elements capable of increasing,stimulating, or enhancing transcription from a basic promoter. The term“silencer”, also referred to herein as “silencer element” is intended toinclude regulatory elements capable of decreasing, inhibiting, orrepressing transcription from a basic promoter. The terms “promoter” and“regulatory element” further encompass “tissue specific” promoters andregulatory elements, i.e., promoters and regulatory elements whicheffect expression of the selected DNA sequence preferentially inspecific cells (e g., cells of a specific tissue). Gene expressionoccurs preferentially in a specific cell if expression in this cell typeis significantly higher than expression in other cell types.

[0042] In some embodiments, one or more copies of a cis-actingregulatory element is present within a transcribed region of a KIM-1gene. In other embodiments, the cis-acting regulatory element is located5′ to the transcriptional start site of a KIM-1 gene.

[0043] In some embodiments, a cis-acting regulatory sequence is operablylinked sequence to a promoter that is not derived from the native KIM-1gene, and to a heterologous sequence, such as a polypeptide-encodingsequence. In other embodiments, the cis-acting regulatory sequence isoperably linked to a KIM-1 promoter sequence and a heterologoussequence, such as a polypeptide-encoding sequence.

[0044] In some embodiments, the cis-acting regulatory sequence includesthe 1.3 kb sequence of SEQ ID NO:3, e.g., the regulatory sequence caninclude the nucleotides of SEQ ID NO:1 and SEQ ID NO:2, as well as SEQID NO:3. In other embodiments, the cis-acting regulatory sequenceincludes a portion of SEQ ID NO:3 that is sufficient to confer renaltissue expression of an operably linked sequence which otherwise wouldnot be expressed in renal tissue. For example, the cis-acting regulatorysequence may include at least 5, 10, 15, 20, 25, 30, 35, 50, 100, 125,or 150 contiguous nucleotides from SEQ ID NO:3, or sequencescomplementary to SEQ ID NO:3. Thus, if desired, sequences responsiblefor conferring renal cell-specific expression in the sequence of SEQ IDNO:3 can be localized more precisely. Localization can be performedusing methods well-known in the art, e.g., by constructing plasmidscontaining successively smaller portions of the 1.3 kb fragment of SEQID NO:3 placed upstream of a luciferase reporter gene in a constructsuch as pGL3 Basic (Promega Corporation, Madison, Wis.), or in any ofthe many reporter genes known in the art. The construct is thentransfected into kidney cells. Suitable kidney cells include, e.g., COS,LLC/PK1, and MDCK cells. Increased expression of the reporter gene inkidney cells compared to the expression of the starting construct aloneindicates that the smaller test fragment of the 1.3 kb DNA allows forrenal tissue expression. Higher expression of the test fragment in renaltissue as compared to other cell types (i.e., fibroblast cells ornon-smooth muscle cells) indicates that the DNA directs polypeptideexpression in a renal tissue-specific manner.

[0045] Similarly, in other embodiments, the cis-acting regulatorysequence is sufficient to confer both inducible (e.g., upon exposure toa stimulus) and tissue-specific expression in injured renal tissue. Forexample, the sequence can include at least the portion or portions ofSEQ ID NO: 2 that are necessary and sufficient for such expression. Suchportion(s) can be localized routinely as described above.

[0046] KIM-1 cis-sequences according to the invention can be used todirect expression of linked sequences following injury or under variousconditions. For example, a nucleotide sequence that includes SEQ ID NO:2(the 4.8 kb KpnI-KpnI fragment) can be used to direct expression of alinked polypeptide in cells that have been subjected to injury using areactive oxygen species (“ROS”), or subjected to injury because ofanoxia. A nucleotide sequence that includes at least the relevantportion or portions of SEQ ID NO:2 can also be used to direct expressionof a sequence of interest in confluent cells.

[0047] In another embodiment, the isolated nucleic acid includes acis-acting KIM-1 derived regulatory sequence that has been modified,e.g., by adding, deleting, or substituting one or more nucleic acidresidues. Such modifications can modulate the regulatory ortranscriptional activity of the regulatory element. For example, amodification can increase or decrease the activity of a promoter orregulatory element. A modification can also affect the tissuespecificity or inducibility of a promoter or regulatory element.

[0048] Desired modifications of a regulatory element according to thepresent invention can be performed according to methods well known inthe art, such as by mutagenesis. The activity of the modified promoteror regulatory element can then be tested, using the herein describedmethods for assaying the cis-acting activity of a KIM-1 regulatorysequence.

[0049] In some embodiments, the regulatory sequence is inducible. Asused herein, “inducible” means that the regulatory sequence affectsexpression of a linked sequence in response to a stimulus. The stimuluscan be physical, e.g., stress, such as heat shock, anoxia, or pressure,or chemical. Examples of chemical stimuli include, e.g., a hormone, acytokine, a heavy metal, phorbol esters, cyclic AMP (cAMP), or retinoicacid. In preferred embodiments, the regulatory sequence is inducible byinjury, e.g., ischemic injury, or ischemia. As used herein, “ischemia”means having a blood flow at least 10% below that which is normal for anindividual of similar size and age, as measured under resting conditionsor exercise conditions. In an adult human, normal resting blood flow isapproximately 1 ml/min/gram of myocardial mass. During exercise, bloodflow typically rises to approximately 3-6 ml/min/gram of myocardialmass. Ischemia may be associated with a physical would or blow, suddenloss of blood volume, toxicity, or a physical obstruction such as atumor.

[0050] Other types of injury include, e.g., injury due to hypertension,chemotherapy (e.g., injury due to cisplatin damage), chronic renalfailure, injury due to auto-immune disorders (e.g., lupus), orpolycystic kidney (PCK) disease.

[0051] In some embodiments, the regulatory sequence preferentiallydirects expression of an operably linked sequence in renal tissue. Theterm “operably linked” means that the regulatory sequence is associatedwith the nucleic acid in such a manner as to facilitate transcription ofthe nucleic acid. In some embodiments, the operably linked nucleic acidencodes an antisense nucleic acid. The antisense nucleic acid can be aportion of the anti-sense strand of a gene whose expression is intendedto be decreased in a renal tissue. For example, for conditionscharacterized by undesired proliferation of kidney tissue, the DNA maybe a KIM-1 antisense nucleic acid.

[0052] In other embodiments, the DNA is operably linked to at least onepolypeptide-encoding sequence. The polypeptide sequence can be, e.g.,one encoded by a KIM-1 cDNA. Examples of nucleic acids encoding rat andhuman KIM-1 cDNAs, and their corresponding encoded amino acid sequencesare provided in PCT publication WO97/44460.

[0053] Alternatively, the DNA is operably linked to nucleic acid thatencodes a polypeptide other than KIM-1. For example, the polypeptide canbe a therapeutic factor such as insulin growth factor (IGF), epidermalgrowth factor (EGF), fibroblast growth factor (FGF), transforming growthfactor beta (TGF β) Type II receptor, particularly the soluble fragmentof TGF β receptor, hepatocyte growth factor (HGF), and the endothelialcell adhesion molecule ICAM-1. Other therapeutic polypeptides includefactors such as a cell survival-promoting factor, a cellgrowth-promoting factor, a wound-healing factor, an anti-fibroticfactor, an apoptosis-inhibiting factor, an anti-inflammatory factor, aterminal differentiation-promoting factor, a cell growth-inhibitingfactor, an intravascular-volume restoration factor, a chelating agent,an alkylating agent, an angiotensin-converting enzyme-inhibiting factor,erythropoietin, a cytokine, a receptor, an anticoagulant, an enzyme, ahormone, an antibody, and a renal structural protein.

[0054] A nucleic acid to be transcribed from a KIM-1 derived regulatoryelement can also be linked to a reporter gene. A reporter gene is anygene encoding a protein, the amount of which can be determined.Exemplary reporter genes include the luciferase gene, e.g., thebacterial luciferase gene, e.g., the luciferase gene present inpGL3-basic (Promega Corp., Madison, Wis.). Other suitable reporter genesinclude the beta-galactosidase gene (LacZ), the chloramphenicol acetyltransferase (CAT) gene, or any gene encoding a protein providingresistance to a specific drug.

[0055] The regulatory elements disclosed herein can also be used toprepare probes and primers based on KIM-1 derived regulatory sequences.These probes and primers can be used, e.g., to identify KIM-1 genomicregions in a subject, such as a human. The probes can be provided in theform of a probe or primer that includes a region of nucleotide sequencethat hybridizes under stringent conditions to at least approximately 6,8, 10 or 12, preferably about 25, 30, 40, 50 or 75 consecutivenucleotides of any of SEQ ID NOS: 1, 2, 3, or 4.

[0056] The probe optionally includes an attached label, which is capableof being detected. The label can be, e.g., radioisotopes, fluorescentmoieties, enzymes, and enzyme co-factors.

[0057] The cis-acting regulatory sequences, including the probe orprimer molecules, can also be used as a part of a diagnostic test kit,for example, to detect mutations in the promoter, which result in faultyexpression of a renal gene or a gene associated with renal tissue.

[0058] Nucleic acids, including nucleic acid fragments, containing orderived from cis-acting KIM-1 regulatory sequences can be preparedaccording to methods well known in the art and described, e.g., inSambrook, J. Fritsch, E. F., and Maniatis, T. (1989) MOLECULAR CLONING:A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. For example, discrete fragments of the regulatory elementcan be prepared and cloned using restriction enzymes. Alternatively,discrete fragments can be prepared using the Polymerase Chain Reaction(PCR) using primers having an appropriate sequence, such as a sequencein SEQ ID NO: 1. The activity of promoter fragments can be tested invitro in transfection assays or in vivo in transgenic animals describedherein. Also within the scope of the invention are nucleic acids thatare homologues or equivalents of the above-described nucleic acids.

[0059] Cis-acting KIM-1 derived regulatory sequences can be isolatedfrom other organisms by using a KIM-1 cDNA to screen genomic DNAsequences in the organism of interest, and then testing the genomicsequences in promoter-reporter assays as described herein. Preferably,the KIM-1 cDNA used for the screening is from the same, or closelyrelated organism. Thus, to isolate a murine KIM-1 derived cis-regulatorysequence, the murine KIM-1 cDNA is used. Preferably, the probe isderived from a 5′ region of the KIM-1 cDNA.

[0060] Vectors and Cells Containing Cis-Acting KIM-1 Derived RegulatorySequences

[0061] This invention also provides vectors, e.g., expression vectorsthat include cis-acting KIM-1 derived regulatory sequences.

[0062] In some embodiments, the expression vector includes a recombinantgene encoding a KIM-1 or a therapeutic polypeptide. Such expressionvectors can be used to transfect cells and thereby produce protein.Constructs containing cis-acting KIM-1 derived regulatory sequences canalso be used as a part of a gene therapy protocol to deliver nucleicacids in vitro or in vivo to particular cell types (e.g., kidney).

[0063] The vector can include any vector known in the art forpropagating a desired nucleic acid in a cell of interest. Thus, thevector can be chosen to propagate a nucleic acid that includes acis-acting KIM-1 derived regulatory sequences in a prokaryotic oreukaryotic host, or both. In some embodiments, the vector is a viralvector, e.g., a retroviral vector. For a review, see Miller, A. D.(1990) Blood 76:271. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound, e.g., in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, F. M.et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14and other standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM.

[0064] Vectors can alternatively be adenovirus-derived vectors, e.g.,those described in Berkner et al. (1988) BioTechniques 6:616; Rosenfeldet al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art.

[0065] The vector can be derived from an adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. Fora review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)158:97-129. It is also one of the few viruses that can integrate its DNAinto non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). See also U.S. Pat. No.5,872,154. Other suitable vectors include those based in the humanimmunodeficiency virus (HIV). These vectors are described in, e.g., U.S.Pat. No. 5,665,577 and U.S. Pat. No. 5,981,276.

[0066] Vectors can be introduced into cells using methods known in theart. In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be used to introduce a gene. Thesemethods include, e.g., calcium phosphate precipitation,microparticle-mediated delivery, and biolistic transformation. In someembodiments, delivery can rely on endocytic pathways for the uptake ofgenes by the targeted cell. Exemplary targeting means of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

[0067] Delivery can be performed using nucleic acids entrapped inliposomes bearing positive charges on their surface (e.g., lipofectins)and (optionally) which are tagged with antibodies against cell surfaceantigens of the target tissue. See, e.g., Mizuno et al. (1992) NoShinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patentapplication 1047381; and European patent publication EP-A-43075. Forexample, lipofection of cells can be carried out using liposomes taggedwith monoclonal antibodies against any cell surface antigen present on ahepatic cell, such as an asialoglycoprotein receptor.

[0068] Cells containing cis-acting KIM-1 regulatory sequences, orvectors that include cis-acting KIM-1 regulatory sequences as describedherein, can be any cell known in the art. Thus, they can includeprokaryotic cells (e.g., E. coli cells) or eukaryotic calls. Eukaryoticcells can include single-celled organisms such, e.g. yeast (e.g.,Saccharomyces cerevisiae or Schizosaccharomyces pombe). Alternatively,the cells can be mammalian cells, e.g., human or simian cells. In someembodiments, the cells are kidney cells, or cell lines derived fromkidney cells.

[0069] Transgenic Animals Containing Cis-Acting KIM-1 RegulatorySequences

[0070] The invention also includes transgenic non-human vertebrates,e.g., mammals and birds, that contain cis-acting KIM-1 regulatorysequences.

[0071] For example, some embodiments, a host cell of the invention is afertilized oocyte or an embryonic stem cell into which exogenouscis-acting KIM-1 regulatory sequences have been introduced. Such hostcells can be used to create non-human transgenic vertebrate animals inwhich exogenous cis-acting KIM-1 regulatory sequences have beenintroduced into their genome or homologous recombinant animals in whichendogenous cis-acting KIM-1 regulatory sequences have been altered. Suchanimals are useful for studying the function and/or activity ofcis-acting KIM-1 regulatory sequences and for identifying and/orevaluating modulators of cis-acting KIM-1 regulatory sequences. As usedherein, a “transgenic animal” means a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, zebrafish, amphibians, etc. A transgene is exogenous DNA thatis integrated into the genome of a cell from which a transgenic animaldevelops and that remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse or a rat, in which an endogenous cis-acting KIM-1regulatory sequences has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

[0072] A transgenic animal of the invention can be created byintroducing cis-acting KIM-1 regulatory sequences into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, or the like, and allowing the oocyte to develop in apseudopregnant female foster animal. For example, a cis-acting KIM-1regulatory sequence having the nucleic acid sequence of SEQ ID NO:3, ora functional fragment thereof, can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of thehuman cis-acting KIM-1 regulatory sequences, such as a cis-acting KIM-1regulatory sequence, can be isolated based on hybridization to the humancis-acting KIM-1 regulatory sequences (described further above) and usedas a transgene. Intronic sequences and polyadenylation signals can alsobe included in the transgene to increase the efficiency of expression ofthe transgene. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, MANIPULATINGTHE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1996). Similar methods are used for production of othertransgenic animals. A transgenic founder animal can be identified basedupon the presence of the transgenic cis-acting KIM-1 derived regulatorysequences in its genome and/or expression of sequences operably linkedto the transgenic cis-acting KIM-1 derived regulatory sequences. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene carrying a transgenic cis-acting KIM-1 derived regulatorysequence can further be bred to other transgenic animals carrying othertransgenes.

[0073] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of a cis-acting KIM-1 regulatorysequences gene into which a deletion, addition or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the cis-actingKIM-1 regulatory sequences. The cis-acting KIM-1 regulatory sequence canbe a human sequence (e.g., SEQ ID NO:3), but more preferably, is anon-human homologue of a human cis-acting KIM-1 regulatory sequence. Forexample, a mouse homologue of human cis-acting KIM-1 regulatory sequenceof SEQ ID NO:3 can be used to construct a homologous recombinationvector suitable for altering a cis-acting KIM-1 regulatory sequence inthe mouse genome.

[0074] In one embodiment, the vector is designed such that, uponhomologous recombination, the endogenous a cis-acting KIM-1 regulatorysequence is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector).

[0075] Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous cis-acting KIM-1 regulatorysequence is mutated or otherwise altered but is still functional (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous cis-acting KIM-1 regulatory sequence). Inthe homologous recombination vector, the altered portion of thecis-acting KIM-1 regulatory sequence is flanked at its 5′ and 3′ ends byadditional nucleic acid of the cis-acting KIM-1 regulatory sequence toallow for homologous recombination to occur between the exogenouscis-acting KIM-1 regulatory sequence carried by the vector and anendogenous cis-acting KIM-1 regulatory sequence in an embryonic stemcell. The additional flanking cis-acting KIM-1 regulatory sequence is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector. See e.g., Thomas el al.(1987) Cell 51:503 for a description of homologous recombinationvectors. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced cis-actingKIM-1 regulatory sequence has homologously recombined with theendogenous cis-acting KIM-1 regulatory sequence are selected (see e.g.,Li et al. (1992) Cell 69:915).

[0076] The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse) to form aggregation chimeras. See e.g., Bradley1987, In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICALAPPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley (1991) Curr Opin Biotechnol 2:823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

[0077] In another embodiment, transgenic non-human animals can beproduced that contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0078] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G₀ phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0079] Identifying Trans-Acting Factors that Bind to Cis-ActingKIM-1-Derived Regulatory Sequences

[0080] Also provided are methods of identifying compounds that bind tocis-acting KIM-1-derived regulatory sequences. These compounds includetrains-acting factors can include, e.g., polypeptides such astranscription factors, which interact preferentially with cis-actingKIM-1 regulatory sequences, or small molecules.

[0081] In one embodiment, a compound is identified by performing assaysin which a cis-acting KIM-1 nucleic acid sequence is incubated with atest compound. Binding of the compound to the nucleic acid is detectedusing methods known in the art for assessing nucleic acid binding. Forexample, binding can be measured using electrophoretic mobility shiftassays (EMSA). One way in which an EMSA can be prepared is to incubatetogether a DNA, which is preferably labeled, containing a KIM-1-derivedcis-acting regulatory sequence and the test compound. The mixture isthen subjected to electrophoresis, and the migration of the labelednucleic acid in the presence of the test compound is compared to themigration of the labeled nucleic acid in the absence of the testcompound. A difference in mobility indicates that the test compoundbinds to regulatory sequence.

[0082] Any suitable compound can be used as the test compound. In someembodiments, the test compound is obtained from a cellular extract knownto contain, or to be suspected of containing, a trans-acting factor.Suitable cells include kidney cells, e.g., Cos cells.

[0083] Cell-based methods can also be used to identify compounds thatmodulate activity. For example, a cell containing a cis-actingKIM-1-derived regulatory sequence operably linked to a nucleic acidencoding a reporter molecule is contacted with a test compound and thereporter molecule mRNA or translated product is measured. mRNA levelsand protein levels can be determined using any method known in the art,e.g. using Northern blot hybridization analysis, immunoprecipitations,or immunohistochemistry.

[0084] The trans-acting factors can also be identified using in vivoassays. For example, a reporter construct can be constructed in which areporter gene is under the control of any of the cis-actingKIM-1-derived regulatory sequences disclosed herein.

[0085] The reporter gene can be any gene encoding a suitably detectableprotein. The reporter gene can be, e.g., a gene encoding luciferase.Cells are transfected with the reporter construct that includes acis-acting KIM-1 regulatory element. Transfection can be transient orstable. The cells can be transfected with more than one reporterconstruct. The transfected cells can then be incubated in the presenceor absence of a test compound for an appropriate amount of time and thelevel of expression of the reporter gene is determined.

[0086] Similar assays can also be performed using a cell or nuclearextract instead of cells. Thus, in one embodiment, the inventionprovides a method for identifying a compound which modulates KIMactivity. The method includes incubating a reporter construct thatincludes any of the regulatory elements according to the invention witha nuclear or cellular extract, or isolated nuclei, in the presence orabsence of test compound. Expression of the test compound is thenmeasured, e.g., by including a labeled nucleotide in the reaction andmeasuring the amount of label incorporated in the product transcribedfrom the reporter construct. Other methods can also be used to determinethe amount of reporter gene expression in this system, such as themeasure of the amount of protein expressed by the reporter gene.

[0087] In yet another embodiment, compounds that modulate the regulatoryelements of the present invention in vivo can be identified in non-humananimals. In one embodiment of the invention, a non-human animal, e.g., amouse, is treated with a compound, such as a compound identified in oneof the assays described above. After an appropriate amount of time, thelevel of activity is determined and compared to its activity in a mousethat has not received the test compound.

[0088] Pharmaceutical Compositions Containing Cis-Acting KIM-1Regulatory Sequences

[0089] Pharmaceutical compositions containing cis-acting KIM-1regulatory sequences can be formulated in a conventional manner usingone or more physiologically acceptable carriers or excipients.

[0090] Administration can be parenteral, intravenous, subcutaneous,intramuscular, retroperitoneal, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, transmucosal, or oral. The Nucleic acids can beprovided in compositions formulated in various ways, according to thecorresponding route of administration. For example, liquid solutions canbe made for ingestion or injection. Gels or powders can be made foringestion, inhalation, or topical application. Methods for making suchformulations are well known and can be found in standard references inthe field, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, MackPublishing Company, Easter, Pa., 15th Edition (1975).

[0091] The compositions can also be formulated as a depot preparation.Such long acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

[0092] The compositions can be presented in a pack or dispenser devicethat may contain one or more unit dosage forms containing the activeingredient. The pack may for example comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration.

[0093] The therapeutic compositions can also contain a carrier orexcipient. Useful excipients include buffers (for example, citratebuffer, phosphate buffer, acetate buffer, and bicarbonate buffer), aminoacids, urea, alcohols, ascorbic acid, phospholipids, proteins (forexample, serum albumin), EDTA, sodium chloride, liposomes, mannitol,sorbitol, and glycerol.

[0094] Methods for Using Cis-Acting KIM-1 Regulatory Sequences

[0095] Cells containing cis-acting KIM-1 regulatory sequences operablylinked to a polypeptide-encoding sequence can be used to directexpression of the polypeptide. For example, the polypeptide can beexpressed by providing a cell containing a KIM-1 regulatory sequence andculturing the cell, if necessary, to allow for expression of thepolypeptide. Any cell type can be used as long as it allows forexpression of the polypeptide-encoding sequence operably linked to thecis-acting regulatory sequence. In preferred embodiments, the cell is arenal cell.

[0096] In some embodiments, the expressed polypeptide is isolated. Ifdesired, the polypeptide-encoding sequence can include a sequenceencoding a signal sequence to allow for secretion of the polypeptide.The polypeptide can then be isolated from the extracellular medium.

[0097] The cis-acting KIM-1 regulatory sequences can also be used toincrease transcription of an operably linked sequence in vitro or invivo, e.g., in a cell, tissue or subject (such as a human). The operablylinked sequence can be, e.g., a polypeptide-encoding sequence or anantisense nucleic acid construct. To increase transcription, a cellcontaining a cis-acting KIM-1 regulatory sequence operably linked to thesequence of interest, or a tissue containing two or more of such cells,is cultured under conditions that allow for the expression of theoperably linked sequence to allow for increased levels of transcriptscorresponding to the operably linked sequence in the cell or tissue.“Culture” as used herein can include in vitro culture under conditionsnecessary for maintaining the viability of mammalian cells, or in situculture of cells in the body of an animal.

[0098] In another embodiment, the cis-acting KIM-1 regulatory sequencesare used to direct expression of a nucleic acid sequence that is notnormally under the control of such regulatory sequences. A nucleic acidmolecule containing a cis-acting KIM-1 regulatory sequence is integratedinto the genome of a target cell in the vicinity of a gene of interest.The gene of interest is preferably one that is normally not expressed inrenal tissue, or is expressed at low amounts in renal tissue.Integration of the introduced cis-acting KIM-1 regulatory sequence nearthe gene of interest allows for the expression of the gene of interestunder the control of the KIM-1 regulatory sequence. Preferably, thecis-acting KIM-1 regulatory sequences are introduced near the 5′ regionof the gene of interest.

[0099] Another use of the cis-acting KIM-1 regulatory sequences is todeliver a therapeutic polypeptide to renal tissue of a subject. Todeliver the polypeptide, a cell including a cis-acting KIM-1 regulatorysequence operably linked to a therapeutic polypeptide-nucleic acidsequence is introduced into renal tissue. The sequences are expressed,e.g., by culturing the cell under conditions that allow for theexpression of the linked polypeptide, to result in the delivery of thetherapeutic polypeptide to the subject's renal tissue. In someembodiments, the therapeutic polypeptide linked to the cis-acting KIM-1regulatory sequence is expressed following a stimulus. The stimulus canbe, e.g., an injury such as ischemic injury or is ischemic reperfusioninjury, or some other nephrotoxic injury.

[0100] The cis-acting KIM-1 regulatory sequences can also be used in amethod for treating or preventing renal tissue injury in a subject. Themethod can include providing a cell in the subject that includes anintroduced cis-acting KIM-1 regulatory sequence operably linked to anucleic acid encoding a therapeutic nucleic acid, e.g., a therapeuticpolypeptide-encoding sequence. The nucleic acid is allowed to express,and the gene product thereby introduced to the renal tissue to preventor treat renal tissue injury in the subject. The cis-acting KIM-1regulatory sequence can be introduced into the subject using methodsdescribed in the art for introducing nucleic acid sequences into cells.The nucleic acids can be introduced ex vivo or in vivo.

[0101] For gene therapy or antisense therapy, the claimed DNA may beintroduced into target cells of an animal, e.g., a patient, usingstandard vectors and/or gene delivery systems. Suitable gene deliverysystems may include liposomes, receptor-mediated delivery systems, nakedDNA, and viral vectors such as herpes viruses, retroviruses,adenoviruses, and adeno-associated viruses, among others. Delivery ofnucleic acids to a specific site in the body for gene therapy orantisense therapy may also be accomplished using a biolistic deliverysystem, such as that described by Williams et al., 1991, Proc. Natl.Acad. Sci. U.S.A. 88:2726-2729. Standard methods for transfecting cellswith isolated DNA are well known to those skilled in the art ofmolecular biology. Gene therapy and antisense therapy to prevent ordecrease the development kidney disease or injury may be carried out bydirectly administering the claimed DNA to a patient or by transfectingrenal cells with the claimed DNA ex vivo and infusing the transfectedcells into the patient.

[0102] A therapeutically effective amount is an amount of the DNA of theinvention that is capable of producing a medically desirable result in atreated animal. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Dosages will vary, but a preferred dosage forintravenous administration of DNA is from approximately 10⁶ to 10²²copies of the DNA molecule.

[0103] Nucleic acids can be delivered to a subject by any of a number ofroutes, e.g., as described in U.S. Pat. Nos. 5,399,346 and 5,580,859.Delivery can thus also include, e.g., intravenous injection, localadministration, and systemic administration (see U.S. Pat. No.5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) PNAS91:3054-3057).

[0104] The pharmaceutical preparation of the gene therapy vector caninclude the gene therapy vector in an acceptable diluent, or can includea slow release matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

EXAMPLES

[0105] The following examples illustrate particular non-limitingembodiments of the invention.

Example 1 Cloning and Characterization of KIM-1 Derived Sequences

[0106] Cis-acting KIM-1 derived regulatory sequences were identified byscreening a human genomic library with a 220 bp Not1-Kpn1 DNA fragmentcontaining sequences in the 5′ region of the human KIM-1 cDNA. The humanKIM-1 cDNA is described generally in PCT publication WO97/44460 andIchimura et al., J. Biol. Chem. 273:4135-42, 1998.

[0107] The screening identified a genomic fragment of 8933 bp (SEQ IDNO:1). The sequence of the 8933 bp region is shown in FIGS. 1A-1H. Atranslational start codon is present beginning at nucleotide 8655.

[0108] A 4817 bp KpnI-KpnI fragment (SEQ ID NO:2), which corresponds tonucleotides 3796-8612 of FIGS. 1A-1H, was subcloned into aluciferase-encoding pGL3b expression vector (Promega Corp.) and namedp4.8KIM/pGL3b. The p4.8KIM/pGL3K construct is shown schematically inFIG. 2B. The presence of the 4817 bp KpnI-KpnI fragment in the pLUC3 wasfound to increase levels of encoded luciferase in renal cells, as isexplained in more detail in the Examples, below.

[0109] A smaller sequence able to increase expression of luciferase inkidney cells was identified by subcloning a 1289 bp EcoRI-KpnI fragment(SEQ ID NO:3) corresponding to nucleotides 7322-8612 in FIG. 1 into apLUC3 vector. The resulting construct was named 1.3 pKIM-1, and is shownschematically in FIG. 2C.

[0110] A construct named 0.5 pKIM, a pLUC3-based construct includingonly nucleotides 8110 to 8612 (SEQ ID NO:4) of FIG. 1, did not increaselevels of luciferase as compared to expression. However, even though theregion 8110 to 8612 is inactive alone, sequences present within theregion may nevertheless be required, along with other sequences, toconfer renal cell expression of linked sequences.

Example 2 Expression in Kidney Cells of Reporter Sequences OperablyLinked to KIM-1 Derived Sequences

[0111] Human genomic sequences from the 5′ region of the human KIM-1gene were tested for their ability to direct expression of a reporterpolypeptide in three kidney-derived cell lines COS cells, a cell linederived from African green monkey kidney fibroblasts; LC/PK cells, acell line derived porcine kidney epithelial cell lines; and MDCK cells,a cell line derived from canine kidney epithelial cells.

[0112] The cell lines were transiently transfected with constructscontaining various regions of DNA from the human KIM-1 gene linked to areporter luciferase gene. These constructs were concomitantlytransfected with pCMV driven β-galactosidase vectors to standardizetransfection efficiency, and activity of luciferase and β-galactosidasewas measured. Activities were calculated as luciferase/β-gal ratios.Relative activities were calculated as the ratio of construct activityto negative control, i.e., promoterless luciferase vector (pGL3b).

[0113] DEAE-mediated transfection was used to introduce constructs(described in more detail below) into cell lines. Transfection wasperformed at 80% confluence, about 24 hours after seeding cells.

[0114] DNA was introduced into the cells by aspirating medium from thecells and mixing 10 ml of the appropriate culture medium (including 10%Nu serum, Collaborative Biomedical Products, #51004), 400 μl of DEAE(1×PBS+10 mg/ml DEAE Dextran+2.5 mM Chloroquine), and 20 μg of DNA (10μg luciferase construct, 2 μg/β-gal vector, 8 μg BlueScript Vector).

[0115] Cells were exposed to DNA for 2-4 hours, after which the DNAsolution was removed by aspiration and replaced with 5 ml 10% DMSO in1×PBS. After 2 minutes, this Solution was removed and the cells werewashed twice with 1×PBS. Fresh medium was added, and cells wereharvested after 48 hours.

[0116] Cells were washed once with 1×PBS, then incubated with 500 μl of1×PBS. Cells were collected and centrifuged for 2 minutes, after whichthe supernatant was removed. Cells were resuspended in 200 μl of 0.25MTRIS, pH 7.8, and subjected to 3 shock freeze-thaw cycles, thencentrifuged for 5 minutes at 14,000 g. The supernatant was used forluciferase and β Galactosidase assays.

[0117] To measure β-galactosidase activity, 25 μl of supernatant, 30 μlof 10×Mg buffer (90 mM MgCl₂. 1.02M beta-mercaptoethanol), 60 μl of 40mM CPRG, and 48 μl of 0.5 sodium phosphate pH 7.5 were mixed andincubated until a red color developed. 500 μl of 1M Na₂CO₃ was added,and the OD at 570 nm was measured.

[0118] To measure luciferase activity 25 μl (p10) of the supernatantwere mixed with 50 μl of 2× assay buffer, as per the manufacturer'sinstructions (Catalog # E 1502, Promega Corporation, Madison, Wis.).Measurements were made using a photoluminometer.

[0119] The KIM-1 human genomic region used to generate the constructs isshown in FIG. 2A. Constructs used in the transfection assays are shownin FIGS. 2B-2D. An 8933 bp fragment (SEQ ID NO:1) is shown schematicallyin FIG. 2A in a 5′ to 3′ orientation. KpnI sites are located atpositions 3796 and 8612 as shown in the figure. For reference, the ATGinitiation codon of the human KIM-1 occurs at position 8655.

[0120] The tested sequences are shown schematically in FIGS. 2B-2D. Onetested sequence included a 4816 bp KpnI-KpnI fragment from the 5′flanking region of the human KIM-1 gene. This fragment corresponds tothe sequences bordered by KpnI sites at positions 3796 and 8612 ofMZ007. FIG. 2B illustrates a construct made by inserting the 4816 bpKpnI-KpnI fragment into a pLUC3 Basic expression vector (PromegaCorporation). The resulting construct was named 4.8 pKIM/PGL3b.

[0121] A shorter fragment from the 4816 bp KpnI-KpnI region was alsotested. This fragment was defined by an EcoRI-KpnI fragment encompassingnucleotides 7322-8611 of MZ007. This KpnI fragment was inserted into thepLUC3 Basic expression vector and named 1.3 pKIM/pGL3b. This constructis shown schematically in FIG. 2C.

[0122] A still shorter fragment defined by a SaclI-KpnI fragmentencompassing nucleotides 8110-8612 of MZ007 was also examined. Thisconstruct was named 0.5 pKIM/pGL3b and is shown schematically in FIG.2D.

[0123] For each cell line tested, relative luciferase activities werecalculated by measuring luciferase following transfection with noplasmid (i.e., zero, “0”), the pGL3 Basic vector alone (“pGL3”), or theconstructs 4.8 pKIM/pGL3b, 1.3 pKIM/pGL3b, and 0.5 pKIM/pGL3b.

[0124] For transformations into COS cells, DMEM was used as the culturemedium. For the 4.8 pKIM construct, four trials were performed, eachusing 2 plates. The RA was 3.8, with SD of 2.82. For the 1.3 pKIMconstruct, four trials were performed, each using 2 plates. The RA was6.3, with an SD of 4.19. For the 0.5 pKIM construct, 2 trials wereperformed, each using 2 plates. The RA as 3.3, with an SD of 1.54. Theresults of the transfection assays using the COS cells are shown in FIG.3.

[0125] For transformations into LLC-PK cells, DMEM was used as theculture medium. For the 4.8 pKIM/PGL3b construct, three trials wereperformed, each using 2 plates. The RA was 5.5, with a SD of 1.43. Forthe 1.3 pKIM/PGL3b construct, three trials were performed, each using 2plates. The RA was 3.4, with an SD of 0.89. For the 0.5 pKIM/PGL3bconstruct, two trials were performed, using 2 plates each. The RA was1.5, with an SD of 0.34. The results of the transfection assays usingLLC-PK cells are summarized in FIG. 4.

[0126] For transformations into MDCK cells, MEM was used as the culturemedium. For the 4.8 pKIM construct, 3 trials were performed, using 2plates each. The relative activity (RA) was 7.0, and the SD was 3.46.For the 1.3 pKIM/PGL3b construct, 3 trials were performed, each using 2plates. The relative activity was 3.4, with a SD of 0.89. The resultsfor the transfection assays using MDCK cells are shown in FIG. 5.

[0127] In assays using COS cells (FIG. 3), LLC/PK1 cells (FIG. 4), andMDCK cells (FIG. 5), luciferase activity was significantly higher incells transfected with the constructs 4.8 pKIM/pGL3b and 1.3 pKIM/pGL3bas compared to cells transfected with pGL3 alone, and cells nottransfected with any construct.

[0128] These results indicate that sequences nucleotides 7322-8612 asshown in FIGS. 1A-H contain cis-acting regulatory elements that are ableto increase expression of operably liked sequences in kidney tissues.Additional elements that increase this activity may be in 3796-7322region, as is shown in FIG. 6, as is discussed in Example 3, below.

[0129] The 0.5 pKIM/pGL3b construct did not increase expression ofluciferase relative to cells transfected with pGL3 alone in the celltypes tested. While these results suggest that these KIM-1 derivedsequences did not confer expression of linked sequences, at least in thecell lines and conditions used, these results do not exclude thepossibility that this region of the KIM-1 flanking region containselements that are necessary or important for increasing expression oflinked sequences in kidney tissues.

Example 3 Expression in Kidney Cells of Reporter Sequences OperablyLinked to KIM-1 Derived Sequences Following Injury

[0130] Expression of luciferase encoded by 4.8 pKIM, 1.3 pKIM, and 0.5pKIM was measured in transfected HK2 cells that had been subjected tochemical anoxia using cyanide and deoxyglucose. HK2 cells are derivedfrom epithelial proximal tubule cells from human kidney.

[0131] A 12-well plate system (MULTIWELL™ 12-well, Becton-Dickson) wasused in these studies. The culture medium used was EGM (Clontech). Priorto transformation, 2 ml medium per well was added to the cells, thenremoved by aspiration. HK2 cells were seeded at a density of30,000/well. Cells reached 80% confluence after 16-24 hours.

[0132] DNA was prepared by mixing 50 μl of serum free medium, 5 μg ofDNA (2.5 μg luciferase construct, 0.5 μg β-galactosidase vector, 2 μgBlueScript vector), and 5 μl of SUPERFECT reagent (Qiagen Corp.), andincubating for 7 minutes. 300 μl of medium+10% FCS was added, and themix was then added to the cells. The cells were then allowed to incubatefor 2 hours. The DNA solution was next removed by aspiration, afterwhich the cells were washed twice in 1×PBS, after which the cells wereincubated in culture medium with 10% FCS.

[0133] To induce chemical anoxia, medium was removed by aspiration, andcells were washed once with 1×PBS. Cells were then incubated for 90minutes in 1 ml Krebs-Henseleit buffer (“KHB”) (6.72 mM NaCl, 3.6 mMKCl, 1.3 mM KH2PO4, 25 mM NaHCO₃, 1 mM CaCl₂, a mM MgCl₂, ph7.4 inincubator), along with 5 mM sodium cyanide and 5 mM deoxyglucose. Cellswere then washed once with 1×PBS and incubated in KHB+10 mM dextrose for15-20 minutes, then harvested as explained below.

[0134] For harvesting, cells were washed once in 1×PBS, then incubatedfor 5 minutes with 200 μl 25 mM GlyGly, 15 mM MgSO4, 4 mM EGTA, pH 8.0,1% Triton X 100, 1 mM DTT. Lysate was then removed from the wells andused for luciferase and galactosidase assays.

[0135] To assay for β galactosidase activity, 50 μl of cell extract wasmixed with 50 μl of assay buffer (Catalog # E2000, Promega Corporation,Madison, Wis.), followed by incubation at 37° C. until a faint yellowcolor developed. Signals were then measured in an ELISA reader at 405nm.

[0136] Luciferase activity was measured by mixing 50 μl of 2× assaybuffer as indicated by the manufacturer (Catalog #E1502, Promega,Madison, Wis.). Measurements were made using a photoluminometer.

[0137] Luciferase expression was measured in three different populationsof transfected HK2 cells. The first group included cells not subjectedto chemical anoxia. These cells were assayed 72 hours followingtransfection. The second group included cells assayed 72 hours followingtransfection and 90 minutes after inducement of chemical anoxia. Foreach group of cells, separate populations of cells were transfected with4.8 pKIM/pGL3b, 1.3 pKIM/GL3b, and 0.5 pKIM/pGL3b.

[0138] The results are illustrated in FIG. 6. For baseline cells, the4.8 pKIM/pGL3b construct yielded a RA of 9.1, with a SEM of 1.1. The 1.3pKIM/pGL3b construct yielded a RA of 5.0, with a SEM of 0.8. The 0.5pKIM constructed yielded a RA of 1.6, with a SEM of 0.2.

[0139] In cells subjected to chemical anoxia, the 4.8 pKIM/pGL3bgenerated a RA of 13.5, with a SEM of 1.5. The 1.3 pKIM/pGL3b constructyielded a RA of 5.6, with a SEM of 0.4. The 0.5 pKIM/pGL3b constructgenerated a RA of 2.0, with a SEM of 0.3.

[0140] These data demonstrate that KIM-1 sequences present in the 4.8pKIM/pGL3b construct caused higher levels of expression of the linkedluciferase gene in cells subjected to anoxia, as compared to controlcells. This effect was not seen with the 1.3 pKIM/pGL3b and 0.5pKIM/pGl3b constructs in this experiment.

Example 4 Expression in Confluent Kidney Cells of Reporter SequencesOperably Linked to KIM-1 Derived Sequences

[0141] Expression of a luciferase gene linked to 4.8 pKIM/PGL3b, 1.3pKIM/PGL3b, or 0.5 pKIM/PGL3b in confluent cells was investigated.

[0142] MDCK cells were transfected with the indicated construct 16-24hours after seeding. Transfected cells were harvested after 24, 48, and72 hours, and luciferase and β-galactosidase activity was then measured.The results are summarized below: TABLE 1 RA SD 24 hours, 80% confluence4.8 pKIM/PGL3b 5.65 0.97 1.3 pKIM/PGL3b 4.93 2.07 0.5 pKIM/PGL3b 2.03.98 48 hours, 90% confluence 4.8 pKIM/PGL3b 13.55 3.5 1.3 pKIM/PGL3b7.95 1.91 0.5 pKIM/PGL3b 2.4 .61 72 hours, 100% confluence 4.8pKIM/PGL3b 31.8 6.5 1.3 pKIM/PGL3b 9.83 4.82 0.5 pKIM/PGL3b 1.93 1.09

[0143] These results demonstrated that KIM-1 sequences present in 4.8pKIM/pGL3B lead to significantly higher levels of luciferase as cellsreach confluency. KIM-1 sequences present only in the 1.3 pKIM/pGL3B and0.5 pKIM/pGL3B constructs did not increase expression of luciferase inthese studies.

Example 5 Use of KIM-1-Derived Cis-Acting Regulatory Sequences in GeneTherapy

[0144] KIM-1 derived cis-acting regulatory sequences according to theinvention can be used for gene therapy treatment of renal diseases.KIM-1 cis-acting regulatory sequences can be used alone or as part of avector to express heterologous genes, e.g., a KIM-1 cDNA, or a proteinother than a KIM-1 polypeptide, in renal cells.

[0145] The DNA or vector containing a KIM-1 cis-acting regulatorysequence linked to a nucleic acid encoding a polypeptide of interest isintroduced into renal cells, which in turn produce the polypeptide ofinterest. For example, sequences encoding the desired polypeptide may beoperably linked to the renal cell-specific promoter sequences of theinvention and expressed in renal cells.

Example 6 Use of KIM-1-Derived Cis-Acting Regulatory Sequences inAntisense Therapy

[0146] The KIM-1 cis-acting regulatory sequence is used in methods ofantisense therapy. Antisense therapy is carried out by administering toan animal, e.g., a human patient, DNA containing the renal cell-specificpromoter sequences of the invention operably linked to a DNA sequence,i.e., an antisense template, which is transcribed into an antisense RNA.The antisense RNA is a short nucleotide sequence (generally at least 10nucleotides, preferably at least 14 nucleotides, and up to 100 or morenucleotides) formulated to be complementary to a portion of a specificmRNA sequence. The antisense template is preferably located downstreamfrom the promoter sequences of the invention. A poly A tail element istypically located at the end of the antisense sequence to signal the endof the sequence. Standard methods relating to antisense technology havebeen described. See, e.g., Melani et al., Cancer Res. 51:2897-2901,1991. Following transcription of the DNA sequence into antisense RNA,the antisense RNA binds to its target mRNA molecules within a cell,thereby inhibiting translation of the mRNA and down-regulatingexpression of the protein encoded by the mRNA.

[0147] The expression of other renal cell proteins may also be inhibitedin a similar manner. For example, the DNA of the invention can beoperably linked to antisense templates that are transcribed intoantisense RNA capable of inhibiting the expression of the followingproteins: TGF-β, dysfunctional collagen mutant genes, WT-1 (Wilms Tumorgene), and genes associated with polycystic kidney disease (PCK).

OTHER EMBODIMENTS

[0148] While the invention has been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the invention, which is defined bythe scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

1 4 1 8933 DNA Homo sapiens 1 gatcatacaa acatgctgtt atttttatcacttaaaaaaa aaacacccag gattttctcc 60 ttccattttt gcaaaacttt tattttttttttggaagatg gggactcact ctgtcactca 120 ggctggaatg cagtagtact accatatctcactgcagcct caaactcctg ggctcaagtg 180 atccctcccg cttagcctcc caaatggctggtactatagg cactcaagtc caactgcttt 240 tctccatgca aactccttga aagtgtttcctgtattcaat tatctcctga ttttccttct 300 tgtaaacttt ttactgcagt ataaagtactggggctcact gataatctcc agcttgctca 360 gtctatgaca aatcttattc ctttcctttgcagcatttga ctcatgattg ctgcctgttc 420 tttgatgcgt ttgcttcact tggcttctaggacctttttg ctttttctct tacctccttg 480 ggctgcttcc atttctgtat tggtgcctcttccacctcag catttttttt tttttttttt 540 tttaagacgg agtctcgctc tctcgcccaggctggagtgc agtggtgcga tctcggctca 600 ctgcaagctc cgcctcccag gttcacgccattctcctgcc tcagcctcct gagtagctgg 660 gactataggc gcccgccacc acgcccggctaatttccacc tcagctttaa caaatttttt 720 taaaattaat taattttttt ttttgagacggagtcttgct ctgtcactca agctggagtg 780 cagtggcatg atctcggctc actgcgacctctgcctccca ggttcaagca attctcctgc 840 ctcagcctcc tgagtagctg ggattacaggcatgcgccat cacacccggc caatttttgt 900 gtttttagta gagacggggt ttcaccatgttggccaggct ggcctggaac tcctgacctc 960 aagtgatcag cctgccttgg tctcctaaagtgctaagact gcaggtgtga gtcgccacac 1020 ccggccttaa aatttattct tatgtagagatggtgtttca ccatgttggc caggctgacc 1080 tggaactcct gaccttaagt tatcagcctgccccggtctc ccaaagtgtt gggattacct 1140 gcatgagtca acatgcttgt ccccattttaatcttttgat gctggaaggc cccaggacct 1200 agtccttagc atcaggcatt cctttgaatctcatcctttg aattcctacc tcattcaggc 1260 tcctggcttt aaaataccat ttttttttttttgaggcgga gtctcgctct gtcgcgcagt 1320 ggcgcgatct cagctcactg caagctccgcctcccaggtt cacaccattc tcctgcctca 1380 gcctcccgag tagctgggac tacaggcacctgccaccacg cctggctaat tttttgtatt 1440 ttcagtagag acggggtttc atcgtgttacccagcacagt ctcgatctcg tgatccgccc 1500 acctcggcct cccaaagtgc tgggattacaggcgtgagcc accgcaccca gccaatacca 1560 tttctaagcc agtaacttgt aactgtatctttagctcaga cctccctcct gaactccagc 1620 agtctccaca caggtctaag acatgtcaaactcaacatac ttaaaaccct gaatatttcc 1680 tctaaaacct gtggtcatgc aggtttttgttttttgtttt ttgttttttt tgagatggag 1740 tcttgctctg ttgcccagac tagagtgcagtgtcacgatc ttggctcact ccaacctctg 1800 cctcctgggt tcaagcaatt ctcctggctcagcctcctga gtagctcaga ttacaggcac 1860 ccacgaccat gcctggctaa atttttgtatttttagtaga gacagggttt tgccatgttg 1920 gccaggttgg tcttgaactc ctgacctcaggtgatccacc tgccttggcc tcccaaggtg 1980 ttaggattac aggtgtgagc cactgagcccagcctttgca gctctccttg tcttaattgg 2040 ctggaacctc cagctcttcc cgtggctcaggccgaaatcc ttggagtcat cttaggccct 2100 ttctcctcat atcctacagg aaatcctgtttgctccacct tctccacctc cttggctcaa 2160 gccattctcc tgcctcagcc tctttagtagctgggactac aagttgcatg ccagcatgcc 2220 tggctaattt ttctttttct ttcttttttttttttttttg tagagacagg gtctcactat 2280 gttgccctga gctcctgggc tcaagcagtcctcccgcctt ggcctcccaa agtccaggga 2340 ttacagctgt gagccatcac atctggctactctaggttga gtgaggaaag ttcattgacc 2400 acttccactg ctaacccatc tcttctggaatctttccata gtctcctgac aggtcttcct 2460 gcttctcaat ctagcaacca cagtggtccttctcaaagga agttagatac tgtcacccta 2520 tgcccttgca gtggtgcttc ttttcatgtggggtgaaagc ctatgtcctc agaatatggc 2580 tcctaagccc catgtgtctg tcctctgccctcactcctct gtgatccctg tccctcgctc 2640 tgttgcagtc acgctggcct ctcttgccctgtaaacacac caggcaccct cctgccttag 2700 ggcctttgcc cttcttgtct gtctccatggaaagcgtttg ctgtcttggc taacttcctt 2760 gtcctttgtc ttagttcaaa taatcaccttcttggtgaaa gtaatagaga ctattcaaac 2820 ctgaccacct tgtttaaaat tgcaactcagtgcctcctca accctccact cccaaccacc 2880 ttcaccctgc tcttgtgtat ccttttgccttttttgcatt agcattcctc aacttgtaat 2940 atgctgataa attacatttt agtgatgttttaaaaatctg tatatttatt tttcagttaa 3000 aagttagtta catgaggcca ggagtggtgctcacgcctat aatcccagca ctttgggagg 3060 ccaaggcggg cagatcactt gaggtcaggagttcgtgacc agcctaacca acatggtgaa 3120 accccgtctc tgctaaaatt acaaaaattagccggtgtgg tgatgcatgc ctgtaatccc 3180 agcttcttgg gaggctgagg taggagaatcgcttgaaccc aggaggcaga gtttgcagtg 3240 agctgagatc gtgccattgc cctccagcctgggcaaaaaa agcgaagctc catctcaaaa 3300 aaaaaaaaaa aaatgtaagt tacatgaggccaggggtctt tggttcattg gtacattcca 3360 gatgaatagg atcatttcta acatatcgcagatcatcaac aaataattgt taaatgagta 3420 cacttttggt atttttatat attttctttctttctttctt tctttctttt tttttttgag 3480 acagaatctc gctctgtcac ccaggctggagtgcagtggt gtgtgatctc agctcactgc 3540 aacctccacc tcccaggttc aagcgattctcttgcctcag cctccctagt atctgagact 3600 acaggcacgc gccaccacgc ctggctaatttttgtagttt tagtagagac aggggtttgc 3660 catattggcc aggctggtct tgaactcctaacctcaagtg atcctcctgc cttggcctcc 3720 caaagtgctg ggattacagg tgtgagccaccatacttggg cttttatgta ttttctatgg 3780 taaacatagg tggtaccctg taatttttatatctttgtaa aagatataaa aaaaagaagc 3840 attatattac ttgttatgaa atcagaggagtaagtgaagg aaaataacta gcttagggca 3900 gtgggcaggg caggaagaga actgaaaggtaggaagacag ttttggaggg aattgcagaa 3960 gtctggatta tagaggccta atataaagtgatggggatga gggagagact gacaggtaca 4020 atgatgtgga gttggtgagt ccctagttgtggagggggcc taagaagatc ttgctgtggt 4080 gaaagcatgg ggaatatgaa cagctgaactgttttgcagg aggctggagc tggaggtacg 4140 atgtgcgctg agatagcagg gaagtaagtggtgattgcaa gaaagaacag tgaattattt 4200 tcttttctga attctttctt ttttttgagacagggtgtca atctgttgtc caggctggag 4260 tgcagtggca cgatctcagc tcactgcaacctccacctcc cgggttcgag caattctcct 4320 gcctcagcct cccaagtagc tgggattacaggcacccacc accgtgcccg gcccatgttc 4380 tgaatcattt caattcactg ccgttaatcttggtttatac agatgcagct ccctagtgag 4440 cagctggaaa ttcagctttg gtgcccaagtattgtcactt ccagctttac cctacaactg 4500 ggatgcatcc ttcagggggg tcatgaagtttgccctaaag agtagtgatc cctggaggtt 4560 gtatagctca ttaaaaaaat ccactgtgctatattgtttg ggagtcttta gaacacaggc 4620 gtctctcatg ggagatggtc ctgtgtcagaaaattcaacc ctatggaatt gtacagttat 4680 gtaacatctc agagccttgg ctccacatccctgtcctggc tctctctggc tcatcatttc 4740 ctccagttga aacaccctcc acccattcttctcacatgtc actttttaag aaattcttcc 4800 caccccccac attccgtcat caaaatgaatggtctttccc tatgggtttg tgtttccatt 4860 tgtttatcta ttcaattaat aacttttttttttttgagaa gtctcactct gtggcccagg 4920 ccagagtgca gtggcatgat ctccgctcagggtaaattct gcctcccggg ttcaggcgat 4980 tctcttgcct cagcctcctg agtagctgggattacaggca cccgccacca cgcctggcta 5040 atttttgcat ttttggtaga gttgggtttcaccatgttgg ccaggctggt ttggaacccc 5100 tgacctcaag tgatcctccc acctcggcctcctttggatt acaggtgtga gcaaccatgc 5160 ctggcttcaa cacttaaatt gccttaaaggagtttatggt ctggagttgg gtgccacaca 5220 acacagtcac tatgtgtgac aatttaaattttattttttt gtttttaatt aatttatttt 5280 tttgaaagct ctgtcatcta aggcttgagtgcagtggtgc catctcaact ccccgaagac 5340 tgtctcctgg gctcaagcaa tctgaaattttaattaaaat gaaattaaat aaaaattttt 5400 aggccaggca tggcggctca cacctgtaattccagcactt ttggaagttg agatgagcgt 5460 atcacttgag gccaggagtt ccagcccagcctggccaaca tggtgaaact ccacctctgc 5520 taaaaataca aaaattagcc aggcatggtggcgcgtgtct gtagtcccag ctactcagga 5580 gactgtggca agagaatcac ttaaacccaggagatggagg ttgcactgag ctgagattgt 5640 gacactgcac tccagcctgg gtgacagagtcaggctctgt cttggaaaaa aaaaaaatta 5700 aaaatgcctt ggttgcctta gccacatttcaagtgctcaa tagtcatatg tggctagtgg 5760 ctgctgtagt gcacgacact cacacagaataactctgtaa ccaatattct actggagaca 5820 gaatcgatcc tatggaattc aaattcaaatcctatggaat tgtacagtta tgtaacatct 5880 cagagcactg gctccacatc cctgtcttggctctctgtgg ctcatcagtt ccagaataac 5940 tccgttacca gaataactcc attactaaaattctaccggg cagcactcta taggagggaa 6000 tagagacaga caccacatat attgcacacacagataaaat ggattaagga aaacaagata 6060 ataatagtga gagggactgg ttggctactttagattgaag gacctgtgaa aaatgtccag 6120 ggaggtcata tttaagccgg gataaaaatgaaaaggaaaa aagtgaaaat ggtggggctg 6180 gggagctaga tggagaacac agccacggaaaaggccttag ggttgaggca agttggaaag 6240 aaagctctag tagctggggc tgagtcagcaggggagagag tggtagaaga aatctatggg 6300 gtaggtcagg gccagaccac cagggcttcagtaatttgag taaagattta ggaattatta 6360 ttattattat tattattatt tttctgagagagttatgaga gggttataag tgggggaatg 6420 atgtagtctg attatatatt tacctttacctcacttatcc tgatttcatt agttgcttac 6480 ttacccatgt ccctgcccga ttgcacaagtctggattttt gacgtcccta gtatattgag 6540 tcatgtccca tcagctcaat atgttagtaataactggttg aattgaatta gctttttttt 6600 ttcaatcttt ttttccttaa gaaacagggtcttgctctgt caccccggct ggtgtgcagt 6660 ggcacaatca tagcctccaa ctgctgggctcaagcaaccc tcctgcctca gcctcctgag 6720 tagctgggac tacggtcagg tacacaaggcctgactatat tttttgttcg tttttttgca 6780 gagagggagt cttgctatgt tgcccaggttggtctcaaac tccttacctc aggtgatcca 6840 cttgccttgg cctcccaaag tgttgggattacaggcgtga gccactgtgc ctggcaagaa 6900 atgaattttt atttttattt ttgagatggagttttgttct tgttgtccag gctagagtgc 6960 aatggcttga tctcggctca ctgcaacctccaccttccag gttcaagcaa ttcttctacc 7020 tcagcctcct aagtagctgg gattacaggcgcccgccacc acccccagct aatttttgta 7080 tttttagtag agtcggggtt tcaccgtgttagccaggctg gtcttgaact cccgacctca 7140 ggtgactggc ctactcggcc tcccaaagtgctggggttac aggcacgagc caccatgccc 7200 ggtcaagaaa tgaattttta aacgctgccatacaaaacac tatgctgaga tcatccactt 7260 ccccatgaac cctgtcatga gctgcaagatacagaccacc actgcctcct tggaagttac 7320 tgaattctta gaccagaaga ggagttaatgaagtactagg caagcttact catgtttgta 7380 tggtttaatg attaacagca gaagtcaacagcccgattta acgcatgtgg gtgcttgaca 7440 cagagcctgc tatatagtat tctccaaaaacctcagctag tgctattact gcatatgatg 7500 taggtttagt tttccaagtt cttccgtggccctttttgct tattatatca atccttggtg 7560 ggagatagag gaagcatttt tagtgctattttacaactga ggaaatagag gtttgaagag 7620 aactcaggaa ctctcagggt tacccagcattgtgagtgac agagcctgga tctgaacgta 7680 agtctgctcc agacttctgt ttcctgaagcattctcttga agtcccttgg taaggaggtg 7740 tagtctgaag catgttgtac aggagcatgaaaggttaggc acagtgattc acattcactc 7800 tcaatttctc ttgctaatgg caaacttggcaatatgactg ttaaggctag ggataagtcg 7860 ttgtggccac tgagtaggaa aagctccacgtccaccagag gcccagttta ctctgaaaag 7920 caagtgcatc tctgccactg gaaggctggcatttgctctc gtgctgccat tgagccacgc 7980 tggttctctg cttccagttt ccttttcttttctttttttt tgttttgttt tttgagacgg 8040 agtcttgctc tgtcgcccag gctggagtgcagtggcgcga tctcggctca ccgcaagctc 8100 cgcctcccgc gggttcacgc cattctcctgcctcagcctc ccgagtagct gggactacag 8160 gcgccagtga ccacgcccgg ctaattttttgtatttttag tagagacggg gtttcaccct 8220 tttagccagg atggtctcga tctcctgacttcgtgatctg cccgccttgg cctcccaaag 8280 tgctaggatt acaggtttga gccaccgcgcccggccctgt ttcctttttg tttgttcccc 8340 tgataccctg tatcaggacc aggagtcagtttggcggtta tgtgtgggga agaagctggg 8400 aagtcagggg ctgtttctgt ggacagctttccctgtcctt tggaaggcac agagctctca 8460 gctgcaggga actaacagag ctctgaagccgttatatgtg gtcttctctc atttccagca 8520 gagcaggctc atatgaatca accaactgggtgaaaagata agttgcaatc tgagatttaa 8580 gacttgatca gataccatct ggtggagggtaccaaccagc ctgtctgctc attttccttc 8640 aggctgatcc cataatgcat cctcaagtggtcatcttaag cctcatccta catctggcag 8700 gtaagtgagt aggtgccctg ggcgggaagaagggagtaga ggggggttag aagccagaga 8760 atggggtagg ggaaggggag gggatggtggtggtggatta atgtagatgt tctttgggta 8820 ccgttgtatg gctatgagtt aactagtgagcaggaccaga ataaagtttt aggccaaaga 8880 aattgcttaa ctgctgtgaa ttacaacattcatggctaaa tgaacaaggc aag 8933 2 4817 DNA Homo sapiens 2 ccctgtaatttttatatctt tgtaaaagat ataaaaaaaa gaagcattat attacttgtt 60 atgaaatcagaggagtaagt gaaggaaaat aactagctta gggcagtggg cagggcagga 120 agagaactgaaaggtaggaa gacagttttg gagggaattg cagaagtctg gattatagag 180 gcctaatataaagtgatggg gatgagggag agactgacag gtacaatgat gtggagttgg 240 tgagtccctagttgtggagg gggcctaaga agatcttgct gtggtgaaag catggggaat 300 atgaacagctgaactgtttt gcaggaggct ggagctggag gtacgatgtg cgctgagata 360 gcagggaagtaagtggtgat tgcaagaaag aacagtgaat tattttcttt tctgaattct 420 ttcttttttttgagacaggg tgtcaatctg ttgtccaggc tggagtgcag tggcacgatc 480 tcagctcactgcaacctcca cctcccgggt tcgagcaatt ctcctgcctc agcctcccaa 540 gtagctgggattacaggcac ccaccaccgt gcccggccca tgttctgaat catttcaatt 600 cactgccgttaatcttggtt tatacagatg cagctcccta gtgagcagct ggaaattcag 660 ctttggtgcccaagtattgt cacttccagc tttaccctac aactgggatg catccttcag 720 gggggtcatgaagtttgccc taaagagtag tgatccctgg aggttgtata gctcattaaa 780 aaaatccactgtgctatatt gtttgggagt ctttagaaca caggcgtctc tcatgggaga 840 tggtcctgtgtcagaaaatt caaccctatg gaattgtaca gttatgtaac atctcagagc 900 cttggctccacatccctgtc ctggctctct ctggctcatc atttcctcca gttgaaacac 960 cctccacccattcttctcac atgtcacttt ttaagaaatt cttcccaccc cccacattcc 1020 gtcatcaaaatgaatggtct ttccctatgg gtttgtgttt ccatttgttt atctattcaa 1080 ttaataactttttttttttt gagaagtctc actctgtggc ccaggccaga gtgcagtggc 1140 atgatctccgctcagggtaa attctgcctc ccgggttcag gcgattctct tgcctcagcc 1200 tcctgagtagctgggattac aggcacccgc caccacgcct ggctaatttt tgcatttttg 1260 gtagagttgggtttcaccat gttggccagg ctggtttgga acccctgacc tcaagtgatc 1320 ctcccacctcggcctccttt ggattacagg tgtgagcaac catgcctggc ttcaacactt 1380 aaattgccttaaaggagttt atggtctgga gttgggtgcc acacaacaca gtcactatgt 1440 gtgacaatttaaattttatt tttttgtttt taattaattt atttttttga aagctctgtc 1500 atctaaggcttgagtgcagt ggtgccatct caactccccg aagactgtct cctgggctca 1560 agcaatctgaaattttaatt aaaatgaaat taaataaaaa tttttaggcc aggcatggcg 1620 gctcacacctgtaattccag cacttttgga agttgagatg agcgtatcac ttgaggccag 1680 gagttccagcccagcctggc caacatggtg aaactccacc tctgctaaaa atacaaaaat 1740 tagccaggcatggtggcgcg tgtctgtagt cccagctact caggagactg tggcaagaga 1800 atcacttaaacccaggagat ggaggttgca ctgagctgag attgtgacac tgcactccag 1860 cctgggtgacagagtcaggc tctgtcttgg aaaaaaaaaa aattaaaaat gccttggttg 1920 ccttagccacatttcaagtg ctcaatagtc atatgtggct agtggctgct gtagtgcacg 1980 acactcacacagaataactc tgtaaccaat attctactgg agacagaatc gatcctatgg 2040 aattcaaattcaaatcctat ggaattgtac agttatgtaa catctcagag cactggctcc 2100 acatccctgtcttggctctc tgtggctcat cagttccaga ataactccgt taccagaata 2160 actccattactaaaattcta ccgggcagca ctctatagga gggaatagag acagacacca 2220 catatattgcacacacagat aaaatggatt aaggaaaaca agataataat agtgagaggg 2280 actggttggctactttagat tgaaggacct gtgaaaaatg tccagggagg tcatatttaa 2340 gccgggataaaaatgaaaag gaaaaaagtg aaaatggtgg ggctggggag ctagatggag 2400 aacacagccacggaaaaggc cttagggttg aggcaagttg gaaagaaagc tctagtagct 2460 ggggctgagtcagcagggga gagagtggta gaagaaatct atggggtagg tcagggccag 2520 accaccagggcttcagtaat ttgagtaaag atttaggaat tattattatt attattatta 2580 ttatttttctgagagagtta tgagagggtt ataagtgggg gaatgatgta gtctgattat 2640 atatttacctttacctcact tatcctgatt tcattagttg cttacttacc catgtccctg 2700 cccgattgcacaagtctgga tttttgacgt ccctagtata ttgagtcatg tcccatcagc 2760 tcaatatgttagtaataact ggttgaattg aattagcttt tttttttcaa tctttttttc 2820 cttaagaaacagggtcttgc tctgtcaccc cggctggtgt gcagtggcac aatcatagcc 2880 tccaactgctgggctcaagc aaccctcctg cctcagcctc ctgagtagct gggactacgg 2940 tcaggtacacaaggcctgac tatatttttt gttcgttttt ttgcagagag ggagtcttgc 3000 tatgttgcccaggttggtct caaactcctt acctcaggtg atccacttgc cttggcctcc 3060 caaagtgttgggattacagg cgtgagccac tgtgcctggc aagaaatgaa tttttatttt 3120 tatttttgagatggagtttt gttcttgttg tccaggctag agtgcaatgg cttgatctcg 3180 gctcactgcaacctccacct tccaggttca agcaattctt ctacctcagc ctcctaagta 3240 gctgggattacaggcgcccg ccaccacccc cagctaattt ttgtattttt agtagagtcg 3300 gggtttcaccgtgttagcca ggctggtctt gaactcccga cctcaggtga ctggcctact 3360 cggcctcccaaagtgctggg gttacaggca cgagccacca tgcccggtca agaaatgaat 3420 ttttaaacgctgccatacaa aacactatgc tgagatcatc cacttcccca tgaaccctgt 3480 catgagctgcaagatacaga ccaccactgc ctccttggaa gttactgaat tcttagacca 3540 gaagaggagttaatgaagta ctaggcaagc ttactcatgt ttgtatggtt taatgattaa 3600 cagcagaagtcaacagcccg atttaacgca tgtgggtgct tgacacagag cctgctatat 3660 agtattctccaaaaacctca gctagtgcta ttactgcata tgatgtaggt ttagttttcc 3720 aagttcttccgtggcccttt ttgcttatta tatcaatcct tggtgggaga tagaggaagc 3780 atttttagtgctattttaca actgaggaaa tagaggtttg aagagaactc aggaactctc 3840 agggttacccagcattgtga gtgacagagc ctggatctga acgtaagtct gctccagact 3900 tctgtttcctgaagcattct cttgaagtcc cttggtaagg aggtgtagtc tgaagcatgt 3960 tgtacaggagcatgaaaggt taggcacagt gattcacatt cactctcaat ttctcttgct 4020 aatggcaaacttggcaatat gactgttaag gctagggata agtcgttgtg gccactgagt 4080 aggaaaagctccacgtccac cagaggccca gtttactctg aaaagcaagt gcatctctgc 4140 cactggaaggctggcatttg ctctcgtgct gccattgagc cacgctggtt ctctgcttcc 4200 agtttccttttcttttcttt ttttttgttt tgttttttga gacggagtct tgctctgtcg 4260 cccaggctggagtgcagtgg cgcgatctcg gctcaccgca agctccgcct cccgcgggtt 4320 cacgccattctcctgcctca gcctcccgag tagctgggac tacaggcgcc agtgaccacg 4380 cccggctaattttttgtatt tttagtagag acggggtttc acccttttag ccaggatggt 4440 ctcgatctcctgacttcgtg atctgcccgc cttggcctcc caaagtgcta ggattacagg 4500 tttgagccaccgcgcccggc cctgtttcct ttttgtttgt tcccctgata ccctgtatca 4560 ggaccaggagtcagtttggc ggttatgtgt ggggaagaag ctgggaagtc aggggctgtt 4620 tctgtggacagctttccctg tcctttggaa ggcacagagc tctcagctgc agggaactaa 4680 cagagctctgaagccgttat atgtggtctt ctctcatttc cagcagagca ggctcatatg 4740 aatcaaccaactgggtgaaa agataagttg caatctgaga tttaagactt gatcagatac 4800 catctggtggagggtac 4817 3 1291 DNA Homo sapiens 3 gaattcttag accagaagag gagttaatgaagtactaggc aagcttactc atgtttgtat 60 ggtttaatga ttaacagcag aagtcaacagcccgatttaa cgcatgtggg tgcttgacac 120 agagcctgct atatagtatt ctccaaaaacctcagctagt gctattactg catatgatgt 180 aggtttagtt ttccaagttc ttccgtggccctttttgctt attatatcaa tccttggtgg 240 gagatagagg aagcattttt agtgctattttacaactgag gaaatagagg tttgaagaga 300 actcaggaac tctcagggtt acccagcattgtgagtgaca gagcctggat ctgaacgtaa 360 gtctgctcca gacttctgtt tcctgaagcattctcttgaa gtcccttggt aaggaggtgt 420 agtctgaagc atgttgtaca ggagcatgaaaggttaggca cagtgattca cattcactct 480 caatttctct tgctaatggc aaacttggcaatatgactgt taaggctagg gataagtcgt 540 tgtggccact gagtaggaaa agctccacgtccaccagagg cccagtttac tctgaaaagc 600 aagtgcatct ctgccactgg aaggctggcatttgctctcg tgctgccatt gagccacgct 660 ggttctctgc ttccagtttc cttttcttttcttttttttt gttttgtttt ttgagacgga 720 gtcttgctct gtcgcccagg ctggagtgcagtggcgcgat ctcggctcac cgcaagctcc 780 gcctcccgcg ggttcacgcc attctcctgcctcagcctcc cgagtagctg ggactacagg 840 cgccagtgac cacgcccggc taattttttgtatttttagt agagacgggg tttcaccctt 900 ttagccagga tggtctcgat ctcctgacttcgtgatctgc ccgccttggc ctcccaaagt 960 gctaggatta caggtttgag ccaccgcgcccggccctgtt tcctttttgt ttgttcccct 1020 gataccctgt atcaggacca ggagtcagtttggcggttat gtgtggggaa gaagctggga 1080 agtcaggggc tgtttctgtg gacagctttccctgtccttt ggaaggcaca gagctctcag 1140 ctgcagggaa ctaacagagc tctgaagccgttatatgtgg tcttctctca tttccagcag 1200 agcaggctca tatgaatcaa ccaactgggtgaaaagataa gttgcaatct gagatttaag 1260 acttgatcag ataccatctg gtggagggta c1291 4 503 DNA Homo sapiens 4 cgggttcacg ccattctcct gcctcagcctcccgagtagc tgggactaca ggcgccagtg 60 accacgcccg gctaattttt tgtatttttagtagagacgg ggtttcaccc ttttagccag 120 gatggtctcg atctcctgac ttcgtgatctgcccgccttg gcctcccaaa gtgctaggat 180 tacaggtttg agccaccgcg cccggccctgtttccttttt gtttgttccc ctgataccct 240 gtatcaggac caggagtcag tttggcggttatgtgtgggg aagaagctgg gaagtcaggg 300 gctgtttctg tggacagctt tccctgtcctttggaaggca cagagctctc agctgcaggg 360 aactaacaga gctctgaagc cgttatatgtggtcttctct catttccagc agagcaggct 420 catatgaatc aaccaactgg gtgaaaagataagttgcaat ctgagattta agacttgatc 480 agataccatc tggtggaggg tac 503

What is claimed is:
 1. An isolated DNA comprising a cis-acting KIM-1derived regulatory sequence.
 2. The DNA of claim 1, wherein said DNAcomprises SEQ ID NO:3.
 3. The DNA of claim 1, wherein said DNA comprisesSEQ ID NO:2.
 4. The DNA of claim 1, wherein said regulatory sequencepreferentially directs expression of an operably linked sequence inrenal tissue.
 5. The DNA of claim 1, wherein said regulatory sequence isinducible.
 6. The DNA of claim 5, wherein said regulatory sequence isinducible by injury.
 7. The DNA of claim 6, wherein said injury isischemic.
 8. The DNA of claim 1, wherein said DNA comprises at least 5contiguous nucleotides from SEQ ID NO:3, or sequences complementary toSEQ ID NO:3.
 9. The DNA of claim 1, wherein said DNA comprises at least5 contiguous nucleotides from a sequence that hybridizes with SEQ IDNO:3, or sequences complementary to SEQ ID NO:3.
 10. The DNA of claim 8,wherein said DNA comprises between 5 and 35 contiguous nucleotides fromSEQ ID NO:3, or sequences complementary to SEQ ID NO:3.
 11. The DNA ofclaim 9, wherein said DNA comprises between 5 and 35 contiguousnucleotides from a sequence that hybridizes with SEQ ID NO:3, orsequences complementary to SEQ ID NO:3.
 12. The DNA of claim 1, whereinsaid DNA is operably linked to a sequence encoding a KIM-1 antisensenucleic acid.
 13. The DNA of claim 1, wherein the DNA is operably linkedto at least one polypeptide-encoding sequence and regulates renaltissue-specific transcription of said polypeptide-encoding sequence 14.The DNA of claim 13, wherein said DNA comprises a portion of SEQ ID NO:3that is sufficient to regulate kidney tissue-specific transcription ofsaid polypeptide-encoding sequence.
 15. The DNA of claim 13, whereinsaid regulatory sequence is inducible.
 16. The DNA of claim 13, whereinsaid polypeptide-encoding sequence encodes a KIM-1 polypeptide.
 17. TheDNA of claim 16, wherein said KIM-1 polypeptide comprises the amino acidsequence of a human KIM-1 polypeptide.
 18. The DNA of claim 13, whereinsaid polypeptide-encoding sequence does not encode a KIM-1 polypeptide.19. The DNA of claim 13, wherein said polypeptide-encoding sequenceencodes a therapeutic polypeptide.
 20. The DNA of claim 13, wherein saidpolypeptide is selected from the group consisting of a cellsurvival-promoting factor, a cell growth-promoting factor, awound-healing factor, an anti-fibrotic factor, an apoptosis-inhibitingfactor, an anti-inflammatory factor, a terminaldifferentiation-promoting factor, a cell growth-inhibiting factor, anintravascular-volume restoration factor, a chelating agent, analkylating agent, an angiotensin-converting enzyme-inhibiting factor,erythropoietin, a cytokine, a receptor, an anticoagulant, an enzyme, ahormone, an antibody, and a renal structural protein.
 21. The DNA ofclaim 13, wherein said polypeptide is selected from the group consistingof an insulin growth factor (IGF), an epidermal growth factor (EGF), afibroblast growth factor (FGF), a transforming growth factor beta (TGFβ) Type II receptor, a hepatocyte growth factor (HGF), and anendothelial cell adhesion molecule ICAM-1.
 22. A vector comprising theDNA of claim
 1. 23. A cell comprising the vector of claim
 22. 24. Thecell of claim 23, wherein said cell is a unicellular organism.
 25. Thecell of claim 23, wherein said cell is a yeast cell.
 26. The cell ofclaim 23, wherein said cell is a mammalian cell.
 27. The cell of claim23, wherein said cell is a human cell.
 28. The cell of claim 23, whereinsaid cell is a non-human mammalian embryonic blastocyst cell.
 29. Atransgenic non-human mammal produced by intrauterine implantation ofsaid blastocyte comprising said cell of claim
 28. 30. One or moreprogeny of said transgenic mammal of claim 29, wherein the DNA of saidprogeny comprises said DNA of claim 1, or a fragment thereof.
 31. Amethod of directing expression of a polypeptide, said method comprising:a) providing a cell comprising the DNA of claim 13; b) culturing saidcell under conditions that allow for the expression of said polypeptide;and c) expressing said polypeptide-encoding sequence; thereby directingexpression of said polypeptide.
 32. The method of claim 31, wherein saidcell is a renal cell.
 33. A method of increasing transcription of apolypeptide-encoding sequence in tissue, said method comprising: a)providing in said tissue a cell comprising the DNA of claim 13; b)culturing said cell under conditions that allow for the transcription ofsaid polypeptide-encoding sequence; and c) expressing saidpolypeptide-encoding sequence; thereby providing increased transcriptionof said polypeptide in said tissue.
 34. A method for identifying a testcompound that modulates expression from a cis-acting KIM-1 derivedregulatory sequence, said method comprising: a) contacting said testcompound and a reporter construct comprised of a reporter gene, operablylinked to said DNA of claim 1; and b) detecting the level of expressionof said reporter gene; wherein a change in the level of expressionrelative to the level of expression in the absence of said test compoundindicates that said test compound modulates the activity of said KIMpromoter.
 35. A method for delivering a therapeutic polypeptide to renaltissue of a subject, said method comprising: a) providing in said renaltissue a cell comprising the DNA of claim 13; b) culturing said cellunder conditions that allow for the expression of said polypeptide; andc) expressing said polypeptide-encoding sequence; thereby deliveringsaid therapeutic polypeptide to said renal tissue of said subject. 36.The method of claim 35, wherein said stimulus is injury.
 37. The methodof claim 36, wherein said injury is an ischemia-reperfusion injury. 38.The method of claim 36, wherein said injury is a nephrotoxic injury. 39.A method for treating or preventing renal tissue injury, the methodcomprising: a) providing a cell comprising the DNA of claim 13; b)culturing said cell under conditions that allow for the expression of atherapeutic polypeptide-encoding sequence; c) expressing saidtherapeutic polypeptide-encoding sequence; and d) contacting said tissuewith said cell expressing said therapeutic polypeptide-encodingsequence; thereby treating or preventing renal tissue injury.
 40. Amethod for increasing transcription of a nucleic acid in a subject, themethod comprising administering to said subject the DNA of claim 4,wherein said operably linked DNA is expressed in an amount sufficient toresult in increased transcription of said operably linked nucleic acid.41. A method for treating or preventing renal tissue injury in asubject, the method comprising administering to said subject in needthereof the DNA of claim 13, wherein said operably linked DNA isexpressed in an amount sufficient to treat or prevent renal tissueinjury in said subject.